The invention relates to a method for transfer of a substance immobilised to magnetic or magnetisable material with the aid of a magnet. The invention is characterised in that the magnet used for transfer is separated from the material to be transferred by an extendable membrane, shapable membrane or by coating of the magnet.
In the traditional method for separating magnetisable material a magnet which is outside the vessel is used. The magnetisable material is left in the vessel and surrounding solution is removed from the vessel.
By introducing the magnet into the solution great advantages are obtained when collecting magnetisable material compared to the traditional methods. It is especially remarkable that in this case the magnetisable material can simply and efficiently be removed from the vessel. When introducing the magnet into the solution the distance of the magnet from the magnetisable material is shorter than when using an outside magnet. Also, due to the fluid""s surface tension collecting magnetisable material left in the solution interface is more efficiently accomplished when the magnet is introduced into the solution.
Patent literature presents numerous devices for separating magnetisable material. The international patent publication WO 87/05536 discloses a separation device within which a permanent magnet moving inside a plastic sleeve can be used to transfer magnetisable material from one vessel to another. The Finnish patent publications (FI 86 05002, FI 95 03669, FI 97 01665, FI 97 01666, FI 97 01667 and FI 97 01668) likewise disclose various methods based on the use of a permanent magnet for transfer of magnetisable material from one vessel to another. The patent publications U.S. Pat. Nos. 4,272,510, 4,649,116 and 4,751,053 disclose magnetic material transfers based on the use of an electromagnet mainly in RIA and EIA assays. The patent publication U.S. Pat. No. 5,567,326 discloses equipment for separation of magnetic particles from the non-magnetic reaction solution with the aid of a steel pin magnetisable with a permanent magnet. Typically, the equipment would include a multiwell reaction plate where magnetic particles can be separated concomitantly in many neighbouring reaction wells using a transfer device with many magnetisable pins. The method described in the patent publication U.S. Pat. No. 5,567,326 is very tedious to use. The unprotected steel pins need to be washed or sterilised in between each time of use. There is a serious risk of contamination in the aforementioned method should the washing not be sufficient.
Magnetic particles have been described in numerous patent publications, for example U.S. Pat. Nos. 3,970,518; 4,018,886; 4,230,685; 4,267,234; 4,452,773; 4,554,088; 4,659,678; 4,978,610; 5,200,084; and 5,705,628. Particle using technology became very popular in for instance immunoassays. Using magnetisable particles for separation of bound antigen-antibody complex from the unbound fraction in immunoassays offered major advantages both in reaction speed and practicality of separation.
Magnetic particles in reaction solution with bound biological material, e.g. cells or an antibody, have been, after the reaction has taken place, secured with the aid of the magnet outside the vessel into a certain location whereupon the solution can be removed without magnetic particles leaving the vessel.
Use of magnetic particles is beneficial because when handling samples, no expensive or space consuming instruments are needed, such as centrifuges, vacuum pumps or chromatographic columns. Magnetic particle applications are simple to perform and volumes used thereupon can vary according to use from small to large.
For the present, magnetic particles are used amongst others in immunoassays, separating cells and bacteria, isolating nucleic acids as well as purification of proteins.
In molecular biology many operations such as isolating and/or transferring nucleic acids as well as using restriction or nucleic acid modifying enzymes pose problems. Among those encountered are inactivation of enzymes, extraction with solvents and star-activity.
Traditionally nucleic acids are isolated and transferred by means of various precipitations and solvent extraction. Some compensatory methods have been presented as aid in nucleic acid management However, these methods are in general expensive and require centrifugation steps. In addition, in some of these methods recovering the nucleic acid in a sufficiently small volume after the operation is difficult.
In methods of molecular biology, where DNA or RNA is manipulated, use is made of restriction enzymes as well as of DNA and/or RNA modifying enzymes. The use of these enzymes is of essential importance in almost all work in the field of molecular biology. The most pre-eminent enzymes in molecular biology labs are the restriction enzymes. These enzymes have made possible major developments in the field. Using restriction enzymes or nucleic acid modifying enzymes in molecular biology applications is mainly routine work which in many cases involves tedious intermittent stages. A good example is provided by the operations needed to eliminate restriction enzyme activity after their use. Many restriction enzymes require phenol extraction in order to inactivate them after use. Phenol extractions are very tedious and from the point of view of the user unpleasant processes. Furthermore, a lot of hazardous waste is generated in these extractions. Commercial manufacturers suggest for many restriction enzymes inactivation by heat treatment whereas in practice users often perform a phenol extraction to insure inactivation of the enzyme. After heat treatment a large percentage of enzyme activity may still remain. Because one has not been able to remove restriction enzymes with currently known techniques the problem has been solved by inactivating enzymes, e.g. by heat or phenol extraction. Another disadvantage is that the used, expensive enzyme can not be reused. Less time consuming but otherwise problematic are various spin columns for purifying DNA from reaction solution. The use of these columns is very expensive, and they are not applicable for removal of many enzymes from DNA solution. Even in this case the retracted enzyme can not be reused.
Phenol extraction is required for inactivation of even many other enzymes commonly used in the field of molecular biology. As examples can be mentioned CIP (Calf Intestinal Phosphatase) and Proteinase K.
No unproblematic means have been presented for transferring and washing restriction enzymes or nucleic acid modifying enzymes. As an example of a problem might be mentioned the star-activity caused by the glycerol used in restriction enzyme storage solution. By this is meant the capacity of restriction enzymes to cut DNA unspecifically, i.e. in places where cutting is not wanted. Commercial restriction enzymes are generally provided as 50% glycerol containing solution. In normal use, a very small amount of restriction enzyme is added to the reaction, even less than 1 xcexcl. If the glycerol content in reaction mixture is too high, it poses, in many cases, a big problem mainly because of the occurrence of star-activity. This sets limits for many molecular biology applications in regard of restriction enzyme use. Another important fact is that it is recommendable to maintain the total volume of the reaction mixture as low as possible in order to have a sufficiently fast enzyme reaction. Commercial restriction enzymes are generally available in one, or at the most two standard concentrations (U/ml). If a great amount of restriction enzyme is wanted in the reaction the glycerol content in reaction solution reaches too high a level. As a result there is star-activity and reaction kinetics are markedly slowed down.
Patent literature suggests preparations in which restriction, or other in molecular biology commonly used, enzymes have been immobilised on a solid support. International patent publication WO 92 15674 suggests immobilising restriction enzymes as well as nucleic acid modifying enzymes onto a surface made of polymer or glass fibre. U.S. Pat. No. 4,342,833 also describes immobilised restriction enzymes using CNBr activated agarose as solid support. On a general level, using magnetic particles in enzyme immobilisation is described in patent publication U.S. Pat. No. 4,698,302 even though in this patent publication there is no mention of enzymes used in the field of molecular biology. In the aforementioned patent publication the separation of magnetic particles was traditionally accomplished with an outside magnet.
In the field of molecular biology capturing of particles poses problems due to the small volumes of fluid in these applications. An acknowledged technique from the fields of cell biology or immunochemistry is not applicable in molecular biology because of the extremely small quantities of liquid used in this field, e.g. 10-100 xcexcl, when corresponding quantities in the field of immunochemistry are several millilitres in magnitude and in the field of cell biology typically 10-100 ml.
Processing cloudy samples or samples containing solid material with magnets traditionally located outside the reaction vessel also poses problems because magnet particles are hard to cleanse of turbidity causing fine particles.
When using traditional outside magnets magnetic particles can not be processed directly transferring them from one reaction vessel to another but have to be processed indirectly by attaching the particles to the reaction vessel wall and changing the surrounding solution with the aid of a pipette.
In the field there is a need for a method that is readily suited for handling small volumes, which is simple to perform, easily automated and readily applicable in various fields.
The purpose of the invention is to accomplish an equipment and a method to ease the handling of target materials that are bound specifically or unspecifically to a magnetic or magnetisable material. In particular, the invention aims to provide easily miniaturised equipment and methods suitable for handling of small sample volumes.
The equipment and method will be used for many widely varying applications such as immunoassays, cell and virus separations, for isolating and purifying nucleic acids as well as for protein purification. The method is particularly suited for isolating, transferring or purifying nucleic acids.
In addition the equipment and method described herein will be used for handling difficult sample materials, such as cloudy samples or samples containing solid material.
Furthermore, the invention strives to accomplish a device for transfer with which magnetic or magnetisable microparticles, including attached thereupon target material, are easily trapped and eventually released. The device for transfer may be of a type that handles simultaneously only one, or more samples. The method may be totalled as a product that comprises the device for transfer with accompanying separation membrane or coating, reagents needed and with which dosage, washing and recapture of magnetic or magnetisable microparticles is easily accomplished.
In particular, the invention aims to provide a method with which the restriction or other enzyme that is used in the field of molecular biology which is immobilised on microparticles can be dosed and transferred from the enzyme containing vessel to the reaction vessel and eventually be removed and recaptured from the reaction vessel. This invention strives amongst other things to increase the ease of use of enzymes used in molecular biological methods and applications and to the reuse of expensive enzymes. By the current method the immobilised enzyme can be washed free of glycerol or other substance interfering with the reaction before it is delivered to the reaction vessel.
The purpose is also to provide a method for protein purification. By the method according to the invention, purifying proteins is markedly easy and proteins may be concentrated at the same time. In protein purification, use can be made of either unspecific or specific protein binding to the magnetic or magnetisable support material.
With this method the dosage of microparticles to a vessel, collecting and transferring them therefrom is easily automated in applications mentioned.
The present invention makes it possible to combine at will procedural steps made up of handling nucleic acids (isolation, transfer, purification) and using bound enzymes as well as allows combination with traditional methods according to the needs of the application at hand.
The characteristics of the invention will emerge from the appended claims. Thus the object of the invention is an equipment and a method for transfer of a microparticle inmmobilised substance from a first vessel to a second vessel. The microparticles are of magnetic or magnetisable material or the microparticles have been attached to a magnetic or magnetisable body. Microparticles with the immobilised substance are collected by aid of a magnet submerged in the first vessel, the magnet along with captured microparticles is transferred to the second vessel and the microparticles are released from the magnet""s influence. It is characteristic of the invention, that the magnet""s surface is separated from the microparticles either with the aid of an extendable membrane or shapable membrane or magnet coating so that the membrane or coating does not essentially weaken the magnetic field directed at the microparticles.
The method according to the invention is especially applicable to fields where small volumes are handled.
In the present application the concept xe2x80x9csubstancexe2x80x9d means any substance immobilised to microparticles that may occur in the application fields of the invention.
xe2x80x9cSubstancexe2x80x9d can therefore stand for e.g. a protein, polypeptide or hapten. The protein can be for example an enzyme, antibody or receptor. The polypeptide can be for example a polypeptide hormone. By hapten are meant low molecular compounds such as lectins, hormones, drugs, pesticides or toxins. Thus also the bioaffinity components used in immunoassays (an antibody or antigen or a complex thereof), for example, and the bioaffinity components used in protein purification (such as a biotinylated protein or streptavidin or a complex thereof) will be included in the concept of xe2x80x9csubstancexe2x80x9d.
xe2x80x9cA substancexe2x80x9d can thus be also a restriction enzyme, modifying enzyme or some other enzyme used in molecular biology, e.g. a protease like proteinase K. As examples of DNA and/or RNA modifying enzymes the following can be mentioned: CIP (Calf Intestinal Phosphatase), Escherichia coli alkaline phosphatase, exonucleases (e.g. P1 nuclease, S1 nuclease), ribonucleases, RNases (e.g. pancreatic RNase RNase H, RNase T1, RNase M, RNase T2), DNA ligases, RNA ligases, DNA polymerases, the Klenow enzyme, RNA polymerases, DNA kinases, RNA kinases, terminal transferases, AMV reverse transcriptase and the phosphodiesterases. The application of these and other DNA and/or RNA modifying enzymes is extremely varied in both research and applications of molecular biology. xe2x80x9cA substancexe2x80x9d can be a nucleic acid, any one or two stranded nucleic acid and especially DNA, RNA, mRNA or cDNA. A nucleic acid can also be PNA (polyamide nucleic acid).
xe2x80x9cA Substancexe2x80x9d can also be a cell such as a T cell, leukocyte, parasite (e.g. Giardia lamblia) and bacterium (e.g. Salmonella sp., E. coli 0157:H7, Listeria monocytogenes, Staphylococcus aureus, Mycobacterium tuberculosis).
xe2x80x9cA substancexe2x80x9d can even be a virus, such as HIV, rotavirus, canine rotavirus, arabis mosaic virus or soybean mosaic virus.
The concept xe2x80x9cmicroparticlexe2x80x9d means in this context rather small particles, preferably in the range of 1.0-10 xcexcm. The microparticle on which a substance is immobilised can be made of a magnetic or magnetisable material. According to another alternative the microparticle itself on which the substance is immobilised can be non-magnetic. In this case the microparticle is suitably attached to another body that is of a magnetic or magnetisable material.
The concept xe2x80x9cimmobilisedxe2x80x9d, when discussing substances immobilised on microparticles, means in this context all such ways, in which the surrounding solution gets in contact with the substance attached to the particle, of attaching or binding the substance, which is to be transferred, to microparticles for the duration of the method of the invention or at least the transfer stages thereof. The immobilised substance can for example be attached on the surface of the particles or it can be captured in a cage like body. xe2x80x9cImmobilisedxe2x80x9d can thus mean also reversible immobilisation in those cases where the substance to be transferred is attached to microparticles for some stages, e.g. transfer stages, and released therefrom at the end of these stages.
xe2x80x9cAttachingxe2x80x9d a substance to microparticles can be accomplished by means of covalent bonding, e.g. making use of the amino or carboxyl groups present on the support. Alternatively, xe2x80x9cattachmentxe2x80x9d can be accomplished using a bioaffinity couple, e.g. biotin/streptavidin couple. One way to proceed is to produce the substance to be immobilised, e.g. an enzyme, by recombinant DNA techniques, e.g. in Escherichia coli bacterial cells, making a special affinity tail on the enzyme. This affinity tail will bind to the microparticles that have suitably attached thereupon some component that will avidly bind the affinity tail in question. The affinity tail may be a low molecular compound, polypeptide or protein. With this arrangement efficient use could be made of microparticles in purifying the desired enzyme and at the same time, the microparticle bound enzyme would be immobilised on the microparticle surface, ready to be used in the method described in the invention.
xe2x80x9cAttachingxe2x80x9d a substance to the microparticles can also be an unspecific non-covalent event such as adsorption. As an example direct attachment of DNA to a glass surface can be mentioned.
The concept of xe2x80x9cmagnetxe2x80x9d, by which the particles are captured, means in this context a material that is either permanently magnetic or that is magnetizable, or a combination of the aforementioned. Ferromagnetic material can suitably be combined with a permanent magnet and/or with an electromagnet. Magnetisation can be carried out either by an electric field or a permanent magnet that is brought into contact with the material to be magnetised. According to the invention, the shape, and size of the magnet may vary.
The concept xe2x80x9cmagnetic or magnetisable materialxe2x80x9d includes paramagnetic, superparamagnetic or ferromagnetic materials. Especially suitable as particle material is any of the superparamagnetic materials. The superparamagnetic particles form for themselves by influence of an outside magnetic field a magnetic field that disappears when the outside magnetic field is removed. Therefore the particles stay separate and do not precipitate which is beneficial to their use. Many commercial manufacturers supply magnetic particles (both paramagnetic and superparamagnetic particles), such as Bangs Laboratories Inc., Dynal A. S., Advanced Magnetics Inc., Scipac Limited, Paesel+Lorei and CPG Inc. Choice can be made amongst differently sized magnetic particles that are activated beforehand and in various ways. Also, magnetic particles that are modified in many different ways are available. As examples can be mentioned magnetic particles that are either carboxy or amino modified. Generally magnetite is bound to a polymeric support such as latex or cellulose. CPG Inc. makes magnetic particles made of porous glass. In all the aforementioned magnetic particles small magnetite crystals (1-20 nm) have been dispersed in polymer and/or glass which is polymerised producing a magnetisable particle. Among others, Prolabo produces magnetic particles that have magnetite in a controllable way only in the core of the particles. This is important, because iron must not be released into the reaction solution in many molecular biological applications such as in a PCR reaction. Iron released in the solution inhibits progress of the reaction in PCR reactions. A magnetic or magnetisable material may also be included in a gel-like substance.
In the method according to the invention the surface of the magnet used is separated from the microparticles by a separate xe2x80x9cmembranexe2x80x9d or xe2x80x9ccoatingxe2x80x9d. xe2x80x9cThe membranexe2x80x9d can be an extendable membrane and/or a shapable membrane. When the magnet is immersed in the vessel in order to capture the microparticles the latter will accumulate to the surface of the membrane or the coating. The magnet along with the microparticles accumulated on the membrane or coating are thereafter taken into a second vessel. In the second vessel the permanent magnet is drawn away from the membranexe2x80x94this will release the microparticles due to the weakened magnetic field. In case of an electromagnet a magnetic field is created for the collecting event and for releasing the magnetic particles the magnetic field is removed. In case of a magnetisable magnet the magnet is magnetised by connecting a permanent magnet to the magnetisable magnet in order to collect and transfer the magnetic particles and the permanent magnet is detached from the magnetisable magnet in order to release the magnetic particles.
The xe2x80x9cmembranexe2x80x9d described in the invention means e.g. a membrane sheet, roll or preshaped membrane. The membrane can suitably have connected thereto reinforcement or support elements for ease of handling. The membrane material can be flexible and/or extendable as long as it can be shaped to suit the magnet used according to the invention. The membrane is preferably thin or it can be made thin by extension. The membrane material is preferably an elastomeric material such as silicone rubber, polyurethane, fluoroelastomer, polychloroprene or chlorosulfonated polyethylen.
The xe2x80x9ccoatingxe2x80x9d described in the invention means a separate surface that is fixed on the magnet. A coating can also be renewed or changed.
An object of the invention is also a device for transfer that is suited for capturing microparticles and releasing the same. The device for transfer is characterised in that the magnet""s surface is separated from the microparticles either by an extendable membrane, shapable membrane or coating so that a membrane or coating, which tightly adheres to the magnet""s surface, separates the magnet from the microparticles but does not essentially weaken the magnetic field targeted at the microparticles.
It is essential that when introducing the device for transfer into the fluid in order to collect the magnetic particles in solution a magnetic field be applied thereon so that the magnetic particles accumulate, due to the magnetic field applied, to the device for transfer. The device for transfer according to the invention can be realised so that the microparticles accumulate suitably on the outer surface of the membrane. The magnet may be designed so that the microparticles will accumulate either on a small area (for example, on the device tip) or on a substantially larger area.
The magnetic particles do not accumulate directly onto the metallic surface of the magnet but around the protective membrane or coating surrounding the magnet. It is most preferred that the protective membrane or coating be an extremely thin protective layer that sits tightly on the magnet or around the same whereby creating in the solution the biggest possible magnetic field force. The protective layer can be a special inert (fixed, permanent) layer of coating forming agent around the magnet, e.g. teflon, silane etc. This is a case that comes in question in particular when using an electromagnet or a magnetisable magnet. The protective membrane can be shapable and/or extendable. In the event of collecting microparticles, the protective membrane may even be extended, thereby decreasing membrane thickness and enforcing the magnetic field. With the device for transfer according to the invention microparticles can be transferred from small volumes of fluid using an extremely small magnet in small vessels.